DNA Replication

Biology I course

Hayder A. Giha

Primary DNA structure

• The primary structure of the DNA is stabilized by 2 types of non-covalent bonds

a. Hydrogen bonds: holds the 2 strands of the DNA together in specific base-pairing manner.

b. Hydrophobic forces: this is a forces between the bases stacking over each other on the same strand.

Eukaryotic DNA synthesis (DNA Replication)

• Replication is synthesis of new DNA from old DNA. • The key enzyme in DNA synthesis is the DNA

polymerase, which selected the nucleotide to be added to the growing strand (based on the corresponding nucleotide in the template strand) and to form the phosphodiester bond between the 5' phosphate of the add nucleotide with the 3' OH of the existing nucleotide.

• The substrates for this enzyme are the deoxy-ribonucleotides triphosphates (dATP, dCTP, dGTP & dTTP) and the single stranded template DNA.

The characteristics of the eukaryotic DNA replication includes

• Semi-conservative with respect to parental strand: during replication one strand of the replicated parent (original or old) DNA is distributed to each of the two daughter (new) DNAmolecules, so the newly synthesized strand together with original strand forms the double strands of daughter DNAs

Characteristics of the eukaryotic DNA replication

• Bidirectional with multiple origins of replication: replication occurs at different sites (origin of replication –O) in one DNA at the same time forming multiple replication forks.

• In each replication fork the replication occurs in the 2 strands but in opposite directions (bidirectional) at the same time.

• The DNA synthesis (replications) occurs from 5' to 3‘ in both strand but in opposite directions

• The DNA is copied at a rate of 50bp/second, so the 3 x 109 bp of the haploid genome are completed in few hours (during S phase of cell cycle).

Characteristics of the eukaryotic DNA replicationPrimed by short stretches of RNA:

• For initiation of DNA replication, a short stretch of RNA need to be added first. The DNA primase (is a DNA-polymerase-associated enzyme) synthesize this short RNA first.

• Chain elongation is done by addition of nucleotides by DNA polymerase to the 3' end of the growing chain

• The selected nucleotide should be complementary to the nucleotide in the template strand (if G in the template the added nucleotide should be C, and if A the added base is T).

• At the end of replication the RNA primers are replaced by short DNA stretches.

Semi-discontinuous• The parental (original) DNA have two stands that run anti-

parallel.• A new strand of DNA is always synthesized in 5' to 3' direction,

the new strand is anti-parallel to the template strand, the latter is being read from 3' to 5' direction.

• Both, the 2 strands of DNA, are read by the same DNA polymerase at the same time but in opposite directions.

• The DNA polymerase synthesized one strand (called leading strand) in the 5' to 3' direction continuously in the same direction of the replication fork movement.

• The other new strand (called lagging strand) is synthesized also from 5' to 3' but discontinuously in a form of small fragments of 100 to 200 bps, known as Okazaki fragments, in the opposite direction of the movement of the fork.

• Each Okazaki fragment is initiated with small primer RNA stretch.• Later on all RNAs are replaced with DNA single strands and

ligated with each other as single strand by DNA ligase.

Proteins involved in DNA synthesizeThe steps of DNA replication includes: i. Defining the origin of replication (multiple origins). ii. Unwinding of the double stranded DNA (loosening the

attachment with histone proteins). iii. Opening of the 2 strand of DNA (breaking H bonds), and

keeping them open. iv. Initiation of replication by addition of RNA primers, v. Polymerization of the deoxy-ribonucleotides. vi. Prevention of torsion (super-coiling) during replication. vii. Termination of the polymerization and putting of the

pieces into one linear DNA (2 daughter DNA molecules). • All these steps need proteins

Proteins involved in DNA synthesize1. DNA polymerases: There are several DNA polymerases (a, b, g, d

and e) found in different locations in the cell and each with distinct function (5' to 3' polymerization, 3' to 5' exonuclease and 5' to 3' exonuclease, proofreading –repair) in DNA replication.

2. Some 3' to 5' exonucleases, are polymerases responsible for proof reading or editing (detection of errors and their corrections).

3. Eukaryotes don’t have 5' to 3' exonuclease that is found in prokaryotes and function in removal of RNA primers.

Proteins involved in DNA synthesize2. DNA helicase: function in unwinding of short segment of parental DNA, (it uses

ATP as source of energy) to separate the 2 strands of DNA (break the H bonds).3. DNA primase: Initiate synthesis of the RNA primers (one in the leading strand and

several in the lagging strand) needed before polymerization of the DNA new strands

4. Single-stranded DNA binding proteins: keeps parental DNA strands separate and protected and prevents premature annealing of the new with the old DNA strands.

5. DNA ligase: catalyze sealing (by phosphodiester bonds) of nicks (breaks) in new DNA backbones after removal of RNA primers and their replacement with DNA.

Proteins involved in DNA synthesize6. Topoisomerases: Rotation of the DNA strands around

one another during replication results in over-wound (super-coiled), this super-twisting can be removed(prevented) by a group of enzymes collectively known as topoisomerases. The enzymes break single strands (nicks), and then reseal the nicks, to release the tension. Types of topoisomerases are:

• Topoisomerase I: catalyze break of single stands of the duplex DNA and rejoining after correcting the supercoils

• Topoisomerase II: catalyze break of both stands of the duplex DNA and rejoining after correcting the supercoils.

Proteins involved in DNA synthesize7. Telomerase: Enzyme help maintain telomeres. The telomere is a

repetitive DNA sequence of TTAGGG, it complexes with proteins at the end of chromosomes.

- Telomeres normally shorten with each cell division and with increasing age.

- Telomeres protect chromosomes from degradation and distinguish broken chromosomes from normal ones.

- The telomerase is RNA-dependent DNA polymerase which add TTAGGG repeats at the end of the chromosomes.

DNA damage• DNA damage can result from both a. endogenous and b. exogenous causes. • Most DNA is repaired before DNA is replicated.• Mutagenic agents (induce mutation) are more damaging during S phase of the

cell cycle when new DNA is synthesized. A. Basal mutation rate: - Is the rate of mutation results from endogenous causes leading to errors in DNA

replication.- Spontaneous changes in the structure of the DNA bases (known as tautomeric

shift) contributes to this type of errors. - However, these faulty bases (unstable forms) are short lived, thus the mutations

due tautomeric shifts are rare.

DNA damage

B. Exogenous agents: Outside factors like; - Ionizing radiation (includes X-ray and radioactive

radiations) are energy rich so can penetrate the whole body and react with DNA and cause both somatic and germ-cells (sex cells) mutations.

- Ultraviolet (UV) radiation is nonionizing and can not penetrate the skin, however, UV radiation from sunlight can be mutagenic and cause skin cancer.

- Some chemicals e.g. hydrocarbons (CH) found in cigarette smoke, are mutagenic. Oxidative free radicalsand chemicals used in chemotherapy for cancer also can induce DNA damage and mutation.

DNA repair system• DNA repair system is needed because the human

body is exposed to several environmental mutagens and also spontaneous mutations occur during DNA replication in each cell.

• Several strategies are adopted by the body to repair the DNA mutations, in most of them undamaged DNA strand used as a template for correction of the errors (mutations).

• When both strands are damaged, the cell uses the sister chromosome (the other copy of the chromosome in diploid cells) as a reference.

• All repair mechanisms use special enzymes that recognize, remove, replace (repair) the faulty base/nucleotide and re-ligate the DNA strand/s.

i. Mismatch repair a. The mismatch repair, corrects the

mismatch between bases in two complementary strands of DNA i.e. failure of the Watson- Crick base pairing (A with T & G with C), a type of errors that occur during replication.

- Such errors – mismatch, are usually recognized by proteins encoded by genes such as; PMS1, PMS2, MSH2, MSH6 & MLH1 genes.

- Mutation in these genes predispose carriers to hereditary colon cancer at young age and other cancers that run in families.

ii. Base-excision repairb. Base-excision repair, corrects

Spontaneous1. de-purination (removal of

purine bases, adenine and guanine), which occurs in a cell at a rate of 10.000 per day

2. De-amination (removal of the amino group from cytosine –which become uracil) of the bases in DNA.

- The repair includes recognitionof the base change and its excision and replacement with the correct base.

iii. Nucleotide excision repair• Used for repair of DNA damage induced

by UV light and some environmental chemicals.

• The UV light is nonionizing, does not penetrate the skin but it induce pyrimidine-pyrimidine dimers between neighboring bases in the DNA. So the UV light from sunlight can results in skin burns and skin cancer.

• This mechanism of repair also used to repair DNA damage induced by chemicals like benzopyrene in cigarette smoke.

• Enzymes of this pathway repair DNA damage by excision of the damaged nucleotides and replace them with normal ones.

Double-stranded DNA repair • Ionizing radiations, oxidative free

radicals, and chemotherapy can damage the 2 strands of the duplex DNA.

• Two repair mechanisms can correct this damage, homologous recombination and non-homologous end joining:

• Homologous recombination: use of sequence information from the unaffected homologous chromosome to repair the damaged one. The proteins BRCA1 and 2, are used in this type of repair. Mutations in the genes coding for BRCA 1 & 2 are associated with breast cancer

Double-stranded DNA repair • Non-homologous end

joining: The process permit joining of ends even if there is no sequence similarity between them, thus, its error-prone repair since it can induce mutation. The repair system is important before DNA replication in the S phase of the cell cycle.

The End